Hepatitis C virus (HCV) infection can result in varied clinical outcomes including acute hepatitis, chronic hepatitis, cirrhosis, or the establishment of an asymptomatic carrier state which may persist for life. It is estimated that HCV establishes chronic infections in >50% of those infected and that about 0.5-1% of the population is infected, with a substantial fraction of these cases without associated risk factors. Recent studies have uncovered a strong association between chronic HCV infection and the development of hepatocellular carcinoma (HCL). Although defining the underlying mechanisms of this association presents a fascinating challenge for cancer researchers, the most direct approaches for reducing the incidence of this disease are 1) to prevent virus infection by developing effective vaccines and 2) to develop therapeutic agents directed against conserved viral targets to control and perhaps cure chronic infections. This proposal is aimed at understanding the molecular mechanisms of HCV replication with emphasis on elucidating viral targets for development of therapeutic agents and vaccines. The first section examines HCV polyprotein processing and virus-encoded enzymatic activities, with emphasis on isolation and characterization of the putative HCV serine proteinase. A byproduct of these studies will be the definition of mature HCV polypeptides produced by proteolysis. This information will allow future studies to be undertaken to assess the involvement of individual HCV products in establishment of chronic infections, virus-induced cytopathic effects, evasion of immunological surveillance, and to determine if these proteins play a direct role in the association of HCV with HCC. The second major focus is to construct full-length HCV cDNA clones and assay transcripts produced from these cloned templates for biological activity. If successful, the establishment of such a system would facilitate screening for effective antivirals, allow molecular genetic studies to further our understanding of HCV replication and host interactions, and might eventually allow engineering of attenuated candidate vaccine strains. The third area of investigation is to develop a novel technology and a set of reagents for studies on T-cell mediated immune responses to HCV infection in primates. This is important for two reasons. First, T-cell immunity may play a role in the ability of some individuals to limit and perhaps eliminate HCV infections. Second, if a set of conserved and protective T-cell epitopes can be identified, such information would aid in the design of potential vaccine strategies and in assessing their efficacy at eliciting appropriate immune responses in vivo.